Protein separation using surfactant precipitation
File(s)
Author(s)
Faizal Wong, Fadzlie Wong
Type
Thesis
Abstract
Protein precipitation using a variety of surfactants has been shown to have considerable potential as a protein separation technique, and considerable work on using anionic surfactants has been carried out by previous researchers. However, anionic surfactants are only suitable for high pI proteins due to concerns about protein stability. Therefore, the first aim of this work was to develop a surfactant precipitation method for low pI proteins based on using cationic surfactants. The effect of important parameters such as the molar ratio of TOMAC to protein (Rp), and pH on the precipitation of bovine serum albumin, α-amylase, and trypsin inhibitor were examined. Recovery of the TOMAC-protein complex by solvent extraction and counter-ionic surfactant (AOT) was also studied. Varied results were obtained for the three proteins, and were correlated with protein properties, and it was found that the protein’s hydrophobicity and molecular weight were the best predictors for precipitation efficiency and recovery.
The second aim of this research was to examine the feasibility of using a biocompatible surfactant – methyl ester sulphonate (MES) as a precipitating-ligand for target proteins in this surfactant precipitation technique. This work was a major breakthrough in the application of a new generation of ‘green’ surfactants for protein extraction. Lysozyme was used as a model protein in a single component system, and the influence of Rp, and pH were examined. Similarly, the recovery of the precipitate using solvent extraction and a counter-ionic surfactant, AOT, was studied. The performance of MES in precipitation was compared to a conventional surfactant, AOT, and it was found that their performance was comparable. This further highlighted its potential to be used as precipitant in protein purification.
The third aim of this work was to apply the surfactant precipitation method to the purification of a target protein from a real industrial sample. Bacteriocin produced by Pediococcus acidilactici Kp10 was chosen as a target protein for this purpose. With a concentration of 11.56 mM of AOT (pH 4), precipitate recovery by acetone (0.99 mM NaCl), and a final recovery phase of 20 mM PBS (pH 7), about 86.3% of overall activity recovery, and a purification factor of about 53.8 was obtained. Further, this separation technique was shown to be better than reverse micellar extraction, and aqueous two-phase extraction in terms of performance. Hence, the surfactant precipitation technique was proven to be an effective and a viable separation method.
The second aim of this research was to examine the feasibility of using a biocompatible surfactant – methyl ester sulphonate (MES) as a precipitating-ligand for target proteins in this surfactant precipitation technique. This work was a major breakthrough in the application of a new generation of ‘green’ surfactants for protein extraction. Lysozyme was used as a model protein in a single component system, and the influence of Rp, and pH were examined. Similarly, the recovery of the precipitate using solvent extraction and a counter-ionic surfactant, AOT, was studied. The performance of MES in precipitation was compared to a conventional surfactant, AOT, and it was found that their performance was comparable. This further highlighted its potential to be used as precipitant in protein purification.
The third aim of this work was to apply the surfactant precipitation method to the purification of a target protein from a real industrial sample. Bacteriocin produced by Pediococcus acidilactici Kp10 was chosen as a target protein for this purpose. With a concentration of 11.56 mM of AOT (pH 4), precipitate recovery by acetone (0.99 mM NaCl), and a final recovery phase of 20 mM PBS (pH 7), about 86.3% of overall activity recovery, and a purification factor of about 53.8 was obtained. Further, this separation technique was shown to be better than reverse micellar extraction, and aqueous two-phase extraction in terms of performance. Hence, the surfactant precipitation technique was proven to be an effective and a viable separation method.
Version
Open Access
Date Issued
2015-08
Date Awarded
2015-12
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
License URL
Advisor
Stuckey, David C.
Ariff, Arbakariya
Sponsor
Malaysia
Publisher Department
Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)